FcγRs belong to the family of Fc receptors (FcRs) which are crucial for defending the human organism against infections. In general, activating FcγRs and inhibiting FcγRs are to be distinguished. Of the three main FcγRs in humans, FcγRI can bind monomeric IgG, whereas FcγRII and FcγRIII bind to multivalent immune complexes (ICs) composed of antibodies and antigens (Takai, T. Nature Reviews Immunology 2002: 580-592.). Effector functions triggered by FcγRs include, depending on the expressed FcR type and associated proteins, endocytosis with subsequent neutralization of the pathogens and antigen presentation, antibody-dependent cellular cytotoxity (ADCC), secretion of mediators or the regulation of antibody production (Fridman et al. Immunol Rev. 1992125:49-76, van de Winkel and Capel Immunol Today. 1993: 14(5):215-21).
WO 00/32767 describes soluble Fc receptors (FcRs) which are composed of only the extracellular part of the receptor and are not glycosylated. Due to the absence of the transmembrane domain and of the signal peptide, these proteins are present in a soluble form and not bound to cells. Furthermore the FcRs described in WO 00/32767 can be produced recombinantly and have been suggested for the treatment of autoimmune diseases due to their ability to bind the Fc part of antibodies without interfering with other components of the immune system. WO 00/32767 additionally describes the crystal structure of certain FcRs and the possibility of finding substances that inhibit the interaction of IgG with FcRs with the aid of these crystal structures. The elucidation of the crystal structure enables the finding of such inhibitors by screening the databases using available computer programs. The invention which as defined in WO 03/043648 further developed the findings of WO 00/32767 and provides treatment methods especially for diseases like multiple sclerosis (MS), systemic lupus erythematosus (SLE), and rheumatoid arthritis (RA) and also for diseases with an elevated level of natural killer cells.
When said receptors were produced recombinantly in prokaryotes and therefore were unglycosylated the inventors of WO 03/043648 surprisingly found that although the unglycosylated proteins were expected to be poorly soluble, the receptors could be purified with high concentrations of FcγR in a soluble form. WO 03/043648 and other publications document that FcRs play an important role in defense reactions of the immune system.
Fc receptors play a central role in the immune system where they control the extent and strength of an immune response. It turned out that in particular a soluble (i.e. the extracellular part of a Fc gamma receptor IIB) Fc gamma receptor IIB (sFcγRIIB), which competes with FcγRs expressed on immune cells for pathogenic immune complexes is beneficial in the treatment of autoimmune diseases. Interference at an early stage of the immune reactions that take place in autoimmune diseases prevents the triggering of the cascade that results in inflammation and tissue destruction. Specifically, meanwhile sFcγRIIB is in phase II clinical trials for the indication Primary Immune Thrombocytopenia (ITP) and Systemic Lupus Erythematosus (SLE). As is commonly known, for clinical trials biological material, here sFcγRIIB is needed that has preferably good Chemistry, Manufacturing and Control (CMC) properties, such as high purity and stability during purification.
Thus it was an object of the present invention to provide human FcγRIIB proteins with good CMC properties. This object is solved by the embodiments reflected in the claims, described herein, illustrated in the Examples and Figures.
Surprisingly it has been shown for the proteins such as those described herein, that higher purification can be achieved due to better solubility at ammonium sulfate concentrations exceeding 1.5M. Ammonium sulfate precipitation is useful to remove large amounts of contaminant proteins, as a first step in many purification schemes. The higher the ammonium sulfate concentration, the better it is when aiming at a highly pure protein, but the more stress is posed upon the protein, because of the high ionic strength of ammonium sulfate. Thus, the more stress resistant a protein is, the higher can be the ammonium sulfate concentration and thus the higher will be the purity of the protein. Specifically, by the addition of the kosmotropic ammonium sulfate byproducts such as unfolded and misfolded species but also host cell derived impurities like cell wall components and proteins are precipitated. With increasing precipitant concentration the precipitation efficiency will be increased and hence a highly purified protein preparation is obtained as long as the protein of interest is resistant to precipitation at such high ammonium sulfate concentrations. As said, it surprisingly turned out that a FcR protein as described herein is highly soluble at ammonium sulfate concentrations equal to or exceeding 1.5 M. This could not have been expected, since prior art FcR proteins behaved differently as is shown in the Examples and there was no guidance whatsoever available as how to modify a FcR protein such that it has the behavior and properties as the FcR protein provided by the present invention. As said, much to the surprise of the present inventors, it turned out that the proteins described herein are indeed resistant to high ammonium sulfate concentrations, thereby allowing a good purification in comparison to prior art FcγRIIB proteins, such as FcγRIIB proteins described in WO 00/32767 or WO 03/043648.